Mycobacterium tuberculosis (Mtb) causes latent infections that affect a third of the world's population and active tuberculosis (TB) kills two million people every year. Chemotherapy of TB requires long treatment regimens and is complicated by the emergence of multi-drug resistant and extensively drug resistant Mtb strains. New drugs that shorten TB chemotherapy and cure drug resistant TB are urgently needed and their development requires a better understanding of the mechanisms used by this pathogen to persist in the host and cause disease. Pathogens need to acquire carbon from the host to establish and maintain an infection. Metabolic pathways used by Mtb during infections are therefore important for pathogenesis and can guide the development of new chemotherapies. Mtb is highly adapted to nutritionally stringent niches in the host and mounting evidence suggests that Mtb preferentially utilizes fatty acids during infections. Our preliminary data indicate that gluconeogenesis is essential for Mtb to grow and persist in immune-competent and immune- compromised mice. We will apply transcriptomic, metabolomic and biochemical approaches to determine the molecular consequences of inhibiting gluconeogenesis in vitro and in vivo, to gain mechanistic insight into the death associated with loss of gluconeogenic enzymes in vitro and during mouse infections, to identify inhibitors of gluconeogenesis and to validate gluconeogenic enzymes as potential drug targets. The proposed work will extend the limited knowledge on Mtb's metabolism during infection and validate novel targets for chemotherapy. PUBLIC HEALTH RELEVANCE: Tuberculosis is one of the world's most devastating diseases. It is responsible for more than two million deaths and eight million new cases annually. Work outlined in this proposal will investigate metabolic adaptations that allow Mycobacterium tuberculosis to grow and persist within its host and to cause disease. It will help validate novel drug targets that might facilitate the development of new drugs against tuberculosis.